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Introduction: Both the quadratus lumborum block (QLB) and the pericapsular nerve group (PENG) block provide effective postoperative analgesia after hip surgery while minimizing the impact on motor function. This study aimed to compare QLB and PENG in patients undergoing primary total hip arthroplasty (THA)., Methods: This superiority trial randomized patients scheduled for elective THA to receive a lateral QLB or a PENG with a lateral femoral cutaneous nerve (LFC) block for postoperative analgesia. Perioperative analgesic protocols were standardized. The primary outcome was postoperative cumulative opioid consumption measured over time up to 72 hours. Secondary outcomes included postoperative pain scores in the first 72 hours, time to ambulation, length of stay, and patient-reported functional outcome measures (Hip disability and Osteoarthritis Outcome Score for Joint Replacement and Patient-Reported Outcome Measures Information System-10 scores)., Results: This trial consented and randomized 106 subjects and 101 were included in the analysis: PENG (n=50), QLB (n=51). Mean (95% CI) opioid consumption in intravenous morphine milligram equivalents differed at 36 hours (mean difference (95% CI), 18.0 (0.80, 35.1); p = 0.040), 48 hours (23.0 (5.20, 40.8); p=0.011), 60 hours (28.0 (9.24, 46.7); p=0.004), and 72 hours (33.0 (13.0, 53.0); p=0.001). There were no significant differences between treatment arms in average resting pain score, time to ambulation, rate of same-day discharge, length of stay, or patient-reported functional outcomes., Conclusion: While both lateral QLB and PENG block+LFC block are effective analgesic methods for patients undergoing THA, patients receiving lateral QLB had decreased cumulative opioid consumption from 36 to 72 hours postoperative and lower pain scores with movement compared with patients receiving PENG+LFC blocks., Trial Registration Number: NCT05710107., Competing Interests: Competing interests: None declared., (© American Society of Regional Anesthesia & Pain Medicine 2024. No commercial re-use. See rights and permissions. Published by BMJ.)

Introduction: The quadratus lumborum block (QLB) and the pericapsular nerve group (PENG) block both provide effective postoperative analgesia after hip surgery while minimizing impact on motor function. This study aimed to compare QLB and PENG in patients undergoing primary total hip arthroplasty., Methods: This superiority trial randomized patients scheduled for elective total hip arthroplasty to receive a lateral QLB or PENG with lateral femoral cutaneous nerve blocks for postoperative analgesia. Perioperative analgesic protocols were standardized. The primary outcome was postoperative cumulative opioid consumption at 72 hours. Secondary outcome was postoperative pain scores. Additional outcomes of interest included time to first ambulation, length of stay, patient reported outcome measures, and opioid-related side effects., Results: This trial consented and randomized 106 subjects and 101 were included in analysis: PENG (n=50), QLB (n=51). Mean (95% CI) opioid consumption (IV MME) in the first 72 hours did not differ between PENG [109.6 (93.6, 125.6)] and QL [92.3 (76.6, 107.9)] groups (p=0.129) There were no significant differences between treatment arms in average pain score, time to ambulation, distance ambulated, rate of same day discharge, or hospital length of stay. There were also no differences in patient reported outcomes using HOOS-JR and PROMIS-10 scores., Conclusion: Patients undergoing primary THA receiving preoperative PENG vs QLB had similar opioid consumption, pain scores, time to ambulation, and hospital length of stay. Both QL and PENG blocks are analgesic options in patients undergoing primary THA., Clinical Trials Registration: NCT05710107; www.ClinicalTrial.gov IRB Protocol ID: Pro00124880., Key Message: Pericapsular nerve group (PENG) block may provide analgesia after hip arthroplasty and improve early functional recovery. This study evaluated postoperative opioid consumption in patients randomized to PENG or lateral quadratus lumborum block (QLB).Opioid consumption, pain scores, motor recovery, and functional outcome measures did not differ in patients randomized to PENG vs lateral QLB.PENG and lateral QLBs are analgesic options following total hip arthroplasty with similar rates of same day discharge.

Nonopioid analgesics serve to improve analgesia and limit side effects and risks of perioperative opioids. N-acetylcysteine (NAC), the primary treatment of acetaminophen toxicity, may have perioperative indications, including analgesia. NAC impacts glutathione synthesis, oxidant scavenging, glutamate receptor modulation and neuroinflammation. Potential perioperative benefits include arrhythmia prevention after cardiac surgery, decreased contrast-induced nephropathy, improved post-transplant liver function and superior pulmonary outcomes with general anesthesia. NAC may improve perioperative analgesia, with some studies displaying a reduction in postoperative opioid use. NAC is generally well tolerated with an established safety profile. NAC administration may predispose to gastrointestinal effects, while parenteral administration may carry a risk of anaphylactoid reactions, including bronchospasm. Larger randomized trials may clarify the impact of NAC on perioperative analgesic outcomes.

Aim: N-acetylcysteine (NAC) decreases inflammation and could augment perioperative analgesia. Materials & methods: This prospective pilot trial examined postoperative opioid consumption at 12 h following intraoperative NAC. In phase I, 20 adults scheduled for posterior spine surgery were randomized to NAC (0, 50, 100 and 150 mg/kg) to determine the optimal dose. In phase II, 30 patients were randomized to placebo or NAC (150 mg/kg). Opioid consumption, pain ratings and time to opioid rescue were recorded. Results: Postoperative opioid consumption was reduced in the NAC group 19.3% at 12 h and 20% at 18 and 36 h. Opioid consumption was reduced 22-24% in the NAC group at all times after adjusting for intraoperative opioid administration. NAC subjects reported lower pain scores relative to placebo. Conclusion: Subjects randomized to NAC consumed less postoperative opioids and reported less pain versus placebo. Larger randomized controlled trials are needed to further evaluate NAC for analgesia. Clinical Trial Registration : NCT04562597 (ClinicalTrials.gov).

Glucagon-like peptide 1 (GLP-1) receptor agonists have surged in popularity for the treatment of both diabetes mellitus and obesity. While GLP-1 reduces proximal gastrointestinal motility and delays gastric emptying, the impact of these medications on adequate fasting before surgery is not clear. We present 2 cases of particulate gastric contents after following appropriate presurgical fasting in diabetic patients taking GLP-1 receptor agonists and review current literature regarding perioperative implications of these drugs., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 International Anesthesia Research Society.)

The National Heart, Lung, and Blood Institute-funded National MDS Natural History Study (NCT02775383) is a prospective cohort study enrolling patients with cytopenia with suspected myelodysplastic syndromes (MDS) to evaluate factors associated with disease. Here, we sequenced 53 genes in bone marrow samples harvested from 1298 patients diagnosed with myeloid malignancy, including MDS and non-MDS myeloid malignancy or alternative marrow conditions with cytopenia based on concordance between independent histopathologic reviews (local, centralized, and tertiary to adjudicate disagreements when needed). We developed a novel 2-stage diagnostic classifier based on mutational profiles in 18 of 53 sequenced genes that were sufficient to best predict a diagnosis of myeloid malignancy and among those with a predicted myeloid malignancy, predict whether they had MDS. The classifier achieved a positive predictive value (PPV) of 0.84 and negative predictive value (NPV) of 0.8 with an area under the receiver operating characteristic curve (AUROC) of 0.85 when classifying patients as having myeloid vs no myeloid malignancy based on variant allele frequencies (VAFs) in 17 genes and a PPV of 0.71 and NPV of 0.64 with an AUROC of 0.73 when classifying patients as having MDS vs non-MDS malignancy based on VAFs in 10 genes. We next assessed how this approach could complement histopathology to improve diagnostic accuracy. For 99 of 139 (71%) patients (PPV of 0.83 and NPV of 0.65) with local and centralized histopathologic disagreement in myeloid vs no myeloid malignancy, the classifier-predicted diagnosis agreed with the tertiary pathology review (considered the internal gold standard)., (Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution.)

Aim: Anesthesia for cardiac surgery has evolved toward fast-track recovery strategies incorporating non opioid analgesics and regional anesthesia. Materials & methods: This retrospective cohort study compared opioid consumption, pain scores and length of stay in patients who underwent cardiac surgery via median sternotomy and did or did not receive preoperative parasternal intercostal plane block catheters with postoperative ropivacaine infusions. Results: Postoperative opioid consumption and postoperative pain scores did not differ. Blocks were associated with decreased intraoperative opioids and reduced length of stay in the intensive care unit and hospital. Conclusion: Parasternal intercostal plane block catheters were not associated with decreased postoperative opioid consumption or pain scores, but were associated with reduced intraoperative opioids and length of stay.

Laser micro-irradiation across the nucleus rapidly generates localized chromatin-associated DNA lesions permitting analysis of repair protein recruitment in living cells. Recruitment of three fluorescently-tagged base excision repair factors [DNA polymerase β (pol β), XRCC1 and PARP1], known to interact with one another, was compared in gene-deleted mouse embryonic fibroblasts and in those expressing the endogenous factor. A low energy micro-irradiation (LEMI) forming direct single-strand breaks and a moderate energy (MEMI) protocol that additionally creates oxidized bases were compared. Quantitative characterization of repair factor recruitment and sensitivity to clinical PARP inhibitors (PARPi) was dependent on the micro-irradiation protocol. PARP1 recruitment was biphasic and generally occurred prior to pol β and XRCC1. After LEMI, but not after MEMI, pol β and XRCC1 recruitment was abolished by the PARPi veliparib. Consistent with this, pol β and XRCC1 recruitment following LEMI was considerably slower in PARP1-deficient cells. Surprisingly, the recruitment half-times and amplitudes for pol β were less affected by PARPi than were XRCC1 after MEMI suggesting there is a XRCC1-independent component for pol β recruitment. After LEMI, but not MEMI, pol β dissociation was more rapid than that of XRCC1. Unexpectedly, PARP1 dissociation was slowed in the absence of XRCC1 as well with a PARPi after LEMI but not MEMI, suggesting that XRCC1 facilitates PARP1 dissociation from specific DNA lesions. XRCC1-deficient cells showed pronounced hypersensitivity to the PARPi talazoparib correlating with its known cytotoxic PARP1 trapping activity. In contrast to DNA methylating agents, PARPi only minimally sensitized pol β and XRCC1-deficient cells to oxidative DNA damage suggesting differential binding of PARP1 to alternate repair intermediates. In summary, pol β, XRCC1, and PARP1 display recruitment kinetics that exhibit correlated and unique properties that depend on the DNA lesion and PARP activity revealing that there are multiple avenues utilized in the repair of chromatin-associated DNA., Competing Interests: Declaration of Competing interest The authors declare there is no conflict of interest. There was no financial or non-financial assistance provided by a third party for the reported work. There are no patents or copyrights relevant to this work., (Published by Elsevier B.V.)

Background: Anaesthesia providers often work at a variety of perioperative and procedural locations. However, the layout of anaesthesia supplies and equipment is often inconsistent from operating theatre to operating theatre. This lack of standardisation may create delays in identification and retrieval of supplies. The primary goal of this study was to compare the duration of time required by anaesthesia providers to identify and retrieve a list of anaesthesia supplies prior to and after standardisation of the location for anaesthesia supplies., Methods: In this observational simulation study, a pre-set list of ten items that may be rapidly needed when caring for patients was created. Volunteer anaesthesia providers were then timed retrieving these in two different operating theatres before and after anaesthesia cart standardisation. Cumulative time to retrieve all items was recorded in seconds. Participants were evaluated regarding mean time to compete the task before and after supply standardisation. Paired t-test were used to assess mean time to retrieve the ten items both before and after standardisation and between the two operating theatre locations. Providers were also evaluated on their familiarity with the operating theatre location, and this was assessed by chi-square tests of homogeneity. Multivariable generalised linear modelling was used to evaluate the impact of covariables on the change in time., Results: Data from 18 anaesthesia providers was collected. Mean (95% confidence interval) time in seconds to retrieve items was decreased by 45% after supply standardisation (105.3 [88.6, 121.9 vs 57.1 [50.8, 63.5]; p < 0.001) with a mean (95% confidence interval) reduction of 48.1 seconds (30.6, 65.6; p < 0.001). Providers who worked primarily at that location also had faster times to complete the task. In a multivariable regression model that considered both the provider's familiarity with the location and the simulation attempt (Operating theatre 1 or Operating theatre 2), the mean time to retrieve all items remained reduced by 48.1 seconds (95% confidence interval: 31.9-64.4) after supply standardisation (p < 0.001)., Conclusion: Standardisation of the location for anaesthesia supplies decreased the time for on-demand item retrieval. Retrieval times were most improved after standardisation for providers in an unfamiliar area. Supply standardisation of anaesthesia carts across perioperative and procedural sites could result in more timely interventions in patient care and efficiency.

Background: Electrical storm can be challenging to treat, requiring a multidisciplinary team to coordinate medical management and invasive procedures. As the stellate ganglion provides efferent sympathetic outflow to the myocardium, stellate ganglion blocks (SGB) can be used to combat ventricular arrhythmias that arise from sympathetic overactivity. Data are scarce regarding SGB catheters as a treatment for electrical storm. We reviewed our use of SGB catheters for refractory electrical storm using our pathway collaboratively developed by critical care, cardiology, and regional anesthesia teams., Methods: We conducted a retrospective cohort study of patients who underwent an SGB for electrical storm between January 2020 and April 2022 in our cardiovascular intensive care unit. The primary outcome was the sustained cessation of electrical storm for 24 hours., Results: Upon chart review, 27 patients were identified and 11 met inclusion criteria. Cessation of electrical storm for 24 hours was achieved in 90% (n=10) of patients after left SGB. Similarly, 90% (n=10) had no documented episodes of ventricular arrhythmias requiring intervention within 6 hours after SGB., Conclusions: SGBs can interrupt or treat electrical storm. SGB catheters allow for prolonged arrhythmia cessation without repeated blocks and decrease the risk of repeat episodes of ventricular arrhythmias., Competing Interests: Competing interests: None declared., (© American Society of Regional Anesthesia & Pain Medicine 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

The double-strand break (DSB) repair pathway called microhomology-mediated end-joining (MMEJ) is thought to be dependent on DNA polymerase theta (Polθ) and occur independently of nonhomologous end-joining (NHEJ) factors. An unresolved question is whether MMEJ is facilitated by a single Polθ-mediated end-joining pathway or consists of additional undiscovered pathways. We find that human X-family Polλ, which functions in NHEJ, additionally exhibits robust MMEJ activity like Polθ. Polλ promotes MMEJ in mammalian cells independently of essential NHEJ factors LIG4/XRCC4 and Polθ, which reveals a distinct Polλ-dependent MMEJ mechanism. X-ray crystallography employing in situ photo-induced DSB formation captured Polλ in the act of stabilizing a microhomology-mediated DNA synapse with incoming nucleotide at 2.0 Å resolution and reveals how Polλ performs replication across a DNA synapse joined by minimal base-pairing. Last, we find that Polλ is semisynthetic lethal with BRCA1 and BRCA2. Together, these studies indicate Polλ MMEJ as a distinct DSB repair mechanism., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Pregnancy is a critical time to provide access to substance use treatment; this is especially true among incarcerated populations, who are known to be at particularly high risk of poor health outcomes. In this integrated literature review, we (1) report what is known about the prevalence of substance use among incarcerated pregnant and postpartum populations; (2) describe substance use treatment programs and current care practices of pregnant and postpartum populations in carceral settings; and (3) explore recommendations and strategies for increasing access to substance use treatment for incarcerated pregnant and postpartum populations. A comprehensive search of seven electronic databases yielded in the retrieval of 139 articles that were assessed for inclusion. Of the retrieved articles, 33 articles met criteria for inclusion in this review. A review of the literature revealed that the understanding of substance use prevalence among pregnant incarcerated women is limited. We also found that treatment of substance use disorders among pregnant and postpartum populations is not routinely available, enhanced perinatal services are sorely needed, and substance use treatment programs are feasible with the help of community partnerships. More research is required to understand current substance use treatment initiatives and outcomes for pregnant women in prison. In addition, strategies for integrating evidence-based, substance use treatment in carceral settings is also needed. Future directions are discussed.

RNA is modified by hundreds of chemical reactions and folds into innumerable shapes. However, the regulatory role of RNA sequence and structure and how dysregulation leads to diseases remain largely unknown. Here, we uncovered a mechanism where RNA abasic sites in R-loops regulate transcription by pausing RNA polymerase II. We found an enhancer RNA, AANCR, that regulates the transcription and expression of apolipoprotein E (APOE). In some human cells such as fibroblasts, AANCR is folded into an R-loop and modified by N-glycosidic cleavage; in this form, AANCR is a partially transcribed nonfunctional enhancer and APOE is not expressed. In contrast, in other cell types including hepatocytes and under stress, AANCR does not form a stable R-loop as its sequence is not modified, so it is transcribed into a full-length enhancer that promotes APOE expression. DNA sequence variants in AANCR are associated significantly with APOE expression and Alzheimer's Disease, thus AANCR is a modifier of Alzheimer's Disease. Besides AANCR, thousands of noncoding RNAs are regulated by abasic sites in R-loops. Together our data reveal the essentiality of the folding and modification of RNA in cellular regulation and demonstrate that dysregulation underlies common complex diseases such as Alzheimer's disease., (Published by Oxford University Press on behalf of Nucleic Acids Research 2022.)

Purpose: The purpose of this study was to examine enhanced perinatal support programs for pregnant and postpartum people in six state prisons, describe the service components offered by each program, and discuss similarities and differences of services offered between programs., Methods: In-depth, semi-structured interviews were conducted with each program's site lead(s) in order to collect information regarding each program's historical context, conception, and key aspects of the implementation of service components offered at each site., Results: Program components fell into five broad categories: group-based education and support, one-on-one support, labor and birth support, lactation facilitation and support, and other support services. Results highlight similarities and differences within and across programs and common themes that govern program success., Conclusions: This study provides an initial understanding of the variation in enhanced perinatal programming in six state prisons and offers insights for other states interested in establishing these types of programs. These programs implemented individual components piecemeal to fit site-specific context and needs, instead of adopting the entirety of another program model. Programs' success was largely dependent upon collaboration between program facilitators and partnering prison sites.

Introduction: Effective analgesia after total hip arthroplasty must minimize pain and optimize early ambulation. Lumbar plexus blocks (LPBs) provide analgesia but may cause motor weakness. Quadratus lumborum blocks (QLBs) may provide analgesia with preserved motor strength., Methods: This trial randomized subjects scheduled for elective hip arthroplasty to receive an LPB or lateral QLB for postoperative analgesia. The primary outcome was opioid consumption at 12-hour postoperative. Non-inferiority of lateral QLBs compared with LPBs was conducted using a one-sided two-sample t-test. Secondary outcomes included pain scores, cumulative opioid consumption, quadriceps strength, time to ambulation, and distance ambulated. Differences in pain scores and opioid consumption over time between groups were evaluated using a linear mixed model., Results: The trial consented and randomized 111 subjects and 103 completed the study: LPB (n=50) and lateral QLB (n=53). Mean (95% CI) cumulative opioid consumption (mg) at 12-hour postoperative was not found to be non-inferior in the lateral QLB (15.9 (12.7 to 19.2)) vs the LPB (12.7 (10.2 to 15.1)) group (p=0.625). Pain scores in postoperative anesthetic care unit (PACU) and 24-hour postoperative did not differ. The maximum distance ambulated did not differ, but lateral QLB patients were 2.4 times more likely to ambulate in the first 12 hours (p=0.024) and had significantly greater quadriceps strength in PACU (p<0.001)., Discussion: Although we were unable to demonstrate non-inferiority for opioid consumption at 12-hour postoperative, strength and mobilization were improved in lateral QLB subjects., Trial Registration Number: NCT04402437., Competing Interests: Competing interests: None declared., (© American Society of Regional Anesthesia & Pain Medicine 2022. No commercial re-use. See rights and permissions. Published by BMJ.)

DNA repair in the context of chromatin is poorly understood. Biochemical studies using nucleosome core particles, the fundamental repeating unit of chromatin, show most DNA repair enzymes remove DNA damage at reduced rates as compared to free DNA. The molecular details on how base excision repair (BER) enzymes recognize and remove DNA damage in nucleosomes have not been elucidated. However, biochemical BER data of nucleosomal substrates suggest the nucleosome presents different structural barriers dependent on the location of the DNA lesion and the enzyme. This indicates the mechanisms employed by these enzymes to remove DNA damage in free DNA may be different than those employed in nucleosomes. Given that the majority of genomic DNA is assembled into nucleosomes, structural information of these complexes is needed. To date, the scientific community lacks detailed protocols to perform technically feasible structural studies of these complexes. Here, we provide two methods to prepare a complex of two genetically fused BER enzymes (Polymerase β and AP Endonuclease1) bound to a single-nucleotide gap near the entry-exit of the nucleosome for cryo-electron microscopy (cryo-EM) structural determination. Both methods of sample preparation are compatible for vitrifying quality grids via plunge freezing. This protocol can be used as a starting point to prepare other nucleosomal complexes with different BER factors, pioneer transcription factors, and chromatin-modifying enzymes.

Management of acute perioperative pain in the geriatric patient can be challenging as the physiologic and pharmacokinetic changes associated with aging may predispose older patients to opioid-related side effects. Furthermore, elderly adults are more susceptible to postoperative delirium and postoperative cognitive dysfunction, which may be exacerbated by both poorly controlled postoperative pain and commonly used pain medications. This narrative review summarizes the literature published in the past 10 years for several nonopioid analgesics commonly prescribed to the geriatric patient in the perioperative period. Nonopioid analgesics are broken down as follows: medications prescribed throughout the perioperative period (acetaminophen and nonsteroidal anti-inflammatory drugs), medications limited to the acute perioperative setting ( N -methyl-D-aspartate receptor antagonists, dexmedetomidine, dexamethasone, and local anesthetics), and medications to be used with caution in the geriatric patient population (gabapentinoids and muscle relaxants). Our search identified 1757 citations, but only 33 specifically focused on geriatric analgesia. Of these, only 21 were randomized clinical trials' and 1 was a systematic review. While guidance in tailoring pain regimens that focus on the use of nonopioid medications in the geriatric patient is lacking, we summarize the current literature and highlight that some nonopioid medications may extend benefits to the geriatric patient beyond analgesia., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 International Anesthesia Research Society.)

Mouse fibroblasts lacking (null) DNA polymerase β (pol β) were transfected with fluorescently tagged pol β and stained with biomarkers to allow visualization within living cells by confocal microscopy. Transient transfection resulted in varying pol β expression levels. Separating cells into three groups based on pol β fluorescence intensity and morphological distribution, permitted analysis of the concentration dependence and spatial distribution of cytoplasmic pol β. Colocalization between pol β and mitochondria was pol β concentration dependent. A decrease in overlap with nucleoids containing mitochondrial DNA (mtDNA) was observed at the highest pol β intensity where pol β exhibits a tubular appearance, suggesting the ability to load elevated levels of pol β into mitochondria readily available for relocation to damaged mtDNA. The dynamics of pol β and mitochondrial nucleoids were followed by confocal recording of time series images. Two populations of mitochondrial nucleoids were observed, with and without pol β. Micro-irradiation, known to form DNA single-strand breaks, in a line across nucleus and cytoplasm of pol β stably transfected cells enhanced apparent localization of pol β with mitochondria in the perinuclear region of the cytoplasm near the nuclear membrane. Exposure of pol β expressing cells to H 2 O 2 resulted in a time-dependent increase in cytoplasmic pol β observed by immunofluorescence analysis of fixed cells. Further screening revealed increased levels of colocalization of pol β with a mitochondrial probe and an increase in oxidative DNA damage in the cytoplasm. ELISA quantification confirmed an increase of an oxidative mitochondrial base lesion, 7,8-dihydro-8-oxoguanine, after H 2 O 2 treatment. Taken together, the results suggest that pol β is recruited to mitochondria in response to oxidatively-induced mtDNA damage to participate in mtDNA repair., (Published by Elsevier B.V.)

Although pectoralis (PECS) blocks are commonly used for breast surgery, recommendations regarding the efficacy of these blocks have thus far not been developed by any professional anesthesia society. Given the potential impact of PECS blocks on analgesia after outpatient breast surgery, The Society for Ambulatory Anesthesia (SAMBA) convened a task force to develop a practice advisory on the use of this analgesic technique. In this practice advisory, we compare the efficacy of PECS blocks with systemic analgesia, local infiltration anesthesia, and paravertebral blockade. Our objectives were to advise on two clinical questions. (1) Does PECS-1 and/or -2 blockade provide more effective analgesia for breast-conserving surgery than either systemic analgesics or surgeon-provided local infiltration anesthesia? (2) Does PECS-1 and/or -2 blockade provide equivalent analgesia for mastectomy compared with a paravertebral block (PVB)? Among patients undergoing breast-conserving surgery, PECS blocks moderately reduce postoperative opioid use, prolong time to analgesic rescue, and decrease postoperative pain scores when compared with systemic analgesics. SAMBA recommends the use of a PECS-1 or -2 blockade in the absence of systemic analgesia (Strength of Recommendation A). No evidence currently exists that strongly favors the use of PECS blocks over surgeon-performed local infiltration in this surgical population. SAMBA cannot recommend PECS blocks over surgical infiltration (Strength of Recommendation C). For patients undergoing a mastectomy, a PECS block may provide an opioid-sparing effect similar to that achieved with PVB; SAMBA recommends the use of a PECS block if a patient is unable to receive a PVB (Strength of Recommendation A)., (© 2022. Society of Surgical Oncology.)

Efficient and accurate DNA synthesis is enabled by DNA polymerase fidelity checkpoints that promote insertion of the right instead of wrong nucleotide. Erroneous X-family polymerase (pol) λ nucleotide insertion leads to genomic instability in double strand break and base-excision repair. Here, time-lapse crystallography captures intermediate catalytic states of pol λ undergoing right and wrong natural nucleotide insertion. The revealed nucleotide sensing mechanism responds to base pair geometry through active site deformation to regulate global polymerase-substrate complex alignment in support of distinct optimal (right) or suboptimal (wrong) reaction pathways. An induced fit during wrong but not right insertion, and associated metal, substrate, side chain and pyrophosphate reaction dynamics modulated nucleotide insertion. A third active site metal hastened right but not wrong insertion and was not essential for DNA synthesis. The previously hidden fidelity checkpoints uncovered reveal fundamental strategies of polymerase DNA repair synthesis in genomic instability., (© 2022. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Background and Objectives: Documentation is important for quality improvement, education, and research. There is currently a lack of recommendations regarding key aspects of documentation in regional anesthesia. The aim of this study was to establish recommendations for documentation in regional anesthesia., Methods: Following the formation of the executive committee and a directed literature review, a long list of potential documentation components was created. A modified Delphi process was then employed to achieve consensus amongst a group of international experts in regional anesthesia. This consisted of 2 rounds of anonymous electronic voting and a final virtual round table discussion with live polling on items not yet excluded or accepted from previous rounds. Progression or exclusion of potential components through the rounds was based on the achievement of strong consensus. Strong consensus was defined as ≥75% agreement and weak consensus as 50%-74% agreement., Results: Seventy-seven collaborators participated in both rounds 1 and 2, while 50 collaborators took part in round 3. In total, experts voted on 83 items and achieved a strong consensus on 51 items, weak consensus on 3 and rejected 29., Conclusion: By means of a modified Delphi process, we have established expert consensus on documentation in regional anesthesia., Competing Interests: Competing interests: None declared., (© American Society of Regional Anesthesia & Pain Medicine 2022. Re-use permitted under CC BY-NC. No commercial re-use. Published by BMJ.)

Compare postoperative opioid consumption following hip arthroscopy in patients who received a preoperative, lateral quadratus lumborum block versus those who did not. Patients were retrospectively identified. The primary outcome was postoperative opioid consumption (IV morphine milligram equivalents) in patients with and without a block. Comparisons between groups were conducted with Chi-square tests and Fisher Exact Tests where appropriate. Continuous data were compared with two-sided t-tests and Wilcoxon rank-sum tests. Data were collected on 100 patients. Mean (95% confidence interval) postoperative opioid consumption was reduced in patients with a quadratus lumborum block compared to those without a block (6.6 [5.2 - 8.1] vs 11.8 [10.2 - 13.4]; p < 0.0001; respectively). Eleven percent of patients with a preoperative quadratus lumborum block did not consume any opioids in the post anesthesia care unit. Opioid consumption in the immediate postoperative period was significantly reduced in patients that received a preoperative quadratus lumborum block. Level of Evidence: Level III - Retrospective Cohort Study. (Journal of Surgical Orthopaedic Advances 31(1):017-021, 2022).

Purpose: To examine acute postoperative opioid consumption in patients undergoing hip arthroscopy and randomized to either receive a preoperative lateral quadratus lumborum block or sham injection., Methods: This trial randomized 46 subjects undergoing hip arthroscopy with a single surgeon to receive a preoperative lateral quadratus lumborum block (40 mL, ropivacaine 0.25%) or sham injection. The primary outcome was postoperative opioid consumption in patients with and without a block. All opioid medications were converted to morphine milligram equivalents for comparisons. Categorical data were compared with χ 2 tests and Fisher exact tests where appropriate. Continuous data were compared with 2-sided t-test and Wilcoxon rank-sum tests., Results: Forty-six subjects scheduled for elective hip arthroscopy were successfully consented and randomized. Demographic and clinical characteristics did not differ. Postoperative opioid consumption decreased 28.3% in patients who received a preoperative lateral quadratus lumborum block (P = .04). Total perioperative opioid consumption (intraoperative and postoperative combined) was reduced 20% in the block group; however, this did not achieve statistical significance (P = .05). Three subjects in the sham group (12.5%) required unblinding for a rescue block in the postoperative anesthetic care unit (PACU) for uncontrolled pain despite systemic analgesics. While cold sensation was decreased postoperatively over the abdomen (P < .001) and anterior thigh (P = .03) in the block group, other PACU variables did not differ, including VAS pain scores, motor function, side effects, PACU duration, and patient satisfaction., Conclusions: Opioid consumption was reduced in patients who received a preoperative lateral quadratus lumborum block combined with a standardized, multimodal protocol as compared with patients who did not receive a block. Our findings support the growing evidence that quadratus lumborum blocks are an effective component of multimodal analgesia options for patients undergoing elective hip arthroscopy., Level of Evidence: Level I, randomized controlled trial., (Copyright © 2021 Arthroscopy Association of North America. Published by Elsevier Inc. All rights reserved.)

Competing Interests: Disclosure The authors have no relevant financial disclosures or conflicts of interest to declare with regards to this article. There is no external funding to report. All authors fully participated in the research, writing, and editing of this article.

Maintaining genome stability involves coordination between different subcellular compartments providing cells with DNA repair systems that safeguard against environmental and endogenous stresses. Organisms produce the chemically reactive molecule formaldehyde as a component of one-carbon metabolism, and cells maintain systems to regulate endogenous levels of formaldehyde under physiological conditions, preventing genotoxicity, among other adverse effects. Dysregulation of formaldehyde is associated with several diseases, including cancer and neurodegenerative disorders. In the present review, we discuss the complex topic of endogenous formaldehyde metabolism and summarize advances in research on fo dysregulation, along with future research perspectives., (Copyright © 2021. Published by Elsevier B.V.)

Lung cancer in never smokers (LCINS) is a common cause of cancer mortality but its genomic landscape is poorly characterized. Here high-coverage whole-genome sequencing of 232 LCINS showed 3 subtypes defined by copy number aberrations. The dominant subtype (piano), which is rare in lung cancer in smokers, features somatic UBA1 mutations, germline AR variants and stem cell-like properties, including low mutational burden, high intratumor heterogeneity, long telomeres, frequent KRAS mutations and slow growth, as suggested by the occurrence of cancer drivers' progenitor cells many years before tumor diagnosis. The other subtypes are characterized by specific amplifications and EGFR mutations (mezzo-forte) and whole-genome doubling (forte). No strong tobacco smoking signatures were detected, even in cases with exposure to secondhand tobacco smoke. Genes within the receptor tyrosine kinase-Ras pathway had distinct impacts on survival; five genomic alterations independently doubled mortality. These findings create avenues for personalized treatment in LCINS., (© 2021. This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply.)

Reactive oxygen species (ROS) oxidize cellular nucleotide pools and cause double strand breaks (DSBs). Non-homologous end-joining (NHEJ) attaches broken chromosomal ends together in mammalian cells. Ribonucleotide insertion by DNA polymerase (pol) μ prepares breaks for end-joining and this is required for successful NHEJ in vivo. We previously showed that pol μ lacks discrimination against oxidized dGTP (8-oxo-dGTP), that can lead to mutagenesis, cancer, aging and human disease. Here we reveal the structural basis for proficient oxidized ribonucleotide (8-oxo-rGTP) incorporation during DSB repair by pol μ. Time-lapse crystallography snapshots of structural intermediates during nucleotide insertion along with computational simulations reveal substrate, metal and side chain dynamics, that allow oxidized ribonucleotides to escape polymerase discrimination checkpoints. Abundant nucleotide pools, combined with inefficient sanitization and repair, implicate pol μ mediated oxidized ribonucleotide insertion as an emerging source of widespread persistent mutagenesis and genomic instability., (© 2021. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Oxidized dGTP (8-oxo-7,8-dihydro-2´-deoxyguanosine triphosphate, 8-oxodGTP) insertion by DNA polymerases strongly promotes cancer and human disease. How DNA polymerases discriminate against oxidized and undamaged nucleotides, especially in error-prone double strand break (DSB) repair, is poorly understood. High-resolution time-lapse X-ray crystallography snapshots of DSB repair polymerase μ undergoing DNA synthesis reveal that a third active site metal promotes insertion of oxidized and undamaged dGTP in the canonical anti-conformation opposite template cytosine. The product metal bridged O8 with product oxygens, and was not observed in the syn-conformation opposite template adenine (A t ). Rotation of A t into the syn-conformation enabled undamaged dGTP misinsertion. Exploiting metal and substrate dynamics in a rigid active site allows 8-oxodGTP to circumvent polymerase fidelity safeguards to promote pro-mutagenic double strand break repair.

Opioid-induced hyperalgesia (OIH) occurs when opioids paradoxically enhance the pain they are prescribed to ameliorate. To address a lack of perioperative awareness, we present an educational review of clinically relevant aspects of the disorder. Although the mechanisms of OIH are thought to primarily involve medullary descending pathways, it is likely multifactorial with several relevant therapeutic targets. We provide a suggested clinical definition and directions for clinical differentiation of OIH from other diagnoses, as this may be confusing but is germane to appropriate management. Finally, we discuss prevention including patient education and analgesic management choices. As prevention may serve as the best treatment, patient risk factors, opioid mitigation, and both pharmacologic and non-pharmacologic strategies are discussed.

Background: Among women with epilepsy, studies regarding changes in seizure frequency during pregnancy have been limited by the lack of an appropriate nonpregnant comparator group to provide data on the natural course of seizure frequency in both groups., Methods: In this prospective, observational, multicenter cohort study, we compared the frequency of seizures during pregnancy through the peripartum period (the first 6 weeks after birth) (epoch 1) with the frequency during the postpartum period (the following 7.5 months after pregnancy) (epoch 2). Nonpregnant women with epilepsy were enrolled as controls and had similar follow-up during an 18-month period. The primary outcome was the percentage of women who had a higher frequency of seizures that impaired awareness during epoch 1 than during epoch 2. We also compared changes in the doses of antiepileptic drugs that were administered in the two groups during the first 9 months of epoch 1., Results: We enrolled 351 pregnant women and 109 controls with epilepsy. Among the 299 pregnant women and 93 controls who had a history of seizures that impaired awareness and who had available data for the two epochs, seizure frequency was higher during epoch 1 than during epoch 2 in 70 pregnant women (23%) and in 23 controls (25%) (odds ratio, 0.93; 95% confidence interval [CI], 0.54 to 1.60). During pregnancy, the dose of an antiepileptic drug was changed at least once in 74% of pregnant women and in 31% of controls (odds ratio, 6.36; 95% CI, 3.82 to 10.59)., Conclusions: Among women with epilepsy, the percentage who had a higher incidence of seizures during pregnancy than during the postpartum period was similar to that in women who were not pregnant during the corresponding epochs. Changes in doses of antiepileptic drugs occurred more frequently in pregnant women than in nonpregnant women during similar time periods. (Funded by the National Institutes of Health; MONEAD ClinicalTrials.gov number, NCT01730170.)., (Copyright © 2020 Massachusetts Medical Society.)

Several cell lines of different origin are routinely used in research and drug development as important models to study human health and disease. Studying cells in culture represents an easy and convenient tool to approach complex biological questions, but the disadvantage is that they may not necessarily reflect what is effectively occurring in vivo. Human primary cells can help address this limitation, as they are isolated directly from human biological samples and can preserve the morphological and functional features of their tissue of origin. In addition, these can offer more relevant data and better solutions to investigators because they are not genetically manipulated. Human foreskin tissue discarded after surgery, for instance, represents a precious source for isolating such cells, including human foreskin fibroblasts (FSK), which are used in several areas of research and medicine. The overall health of cells is determined by the mitochondria. Alterations of cellular metabolism and cell death pathways depend, in part, on the number, size, distribution, and structure of mitochondria, and these can change under different cellular and pathological conditions. This highlights the need to develop accurate approaches to study mitochondria and evaluate their function. Here, we describe three easy, step-by-step protocols to study cellular viability and mitochondrial functionality in FSK. We describe how to use circumcision tissue obtained from the clinic to isolate FSK cells by mechanical and enzymatic disaggregation, how to use a cationic dye, crystal violet, which is retained by proliferating cells, to determine cell viability, and how to prepare samples to assess the metabolic status of cells by evaluating different mitochondrial parameters with transmission electron microscopy. We have successfully used the approaches outlined here to recapitulate physiological conditions in these cells in order to study the effects of increased intracellular levels of formaldehyde. © 2020 U.S. Government. Basic Protocol 1: Isolation and maintenance of human primary foreskin fibroblasts (FSK) Basic Protocol 2: Determination of cell viability by crystal violet staining Basic Protocol 3: Transmission electron microscopy to study cellular damage and mitochondrial dysfunction., (Published 2020. This article is a US Government work and is in the public domain in the USA.)

Background: Preoperative screening and testing for SARS-CoV-2 are important aspects of reopening perioperative and procedural sites to elective cases after the initial wave of the novel coronavirus pandemic. However, given that modern healthcare has never experienced a pandemic of this magnitude, rapid operationalization of mass testing presents unique challenges. We aim to highlight our experiences and challenges for preoperative SARS-CoV-2 testing., Methods: We describe implementation of widespread screening tools and preoperative polymerase chain reaction (PCR) testing in a single, academic medical center., Results: As of August 2020, we have been able to achieve an over 90% success rate in preoperative SARS-CoV-2 PCR testing for both outpatient and inpatient procedures. However, there are certain challenges in obtaining high levels of compliance both on individual and institutional levels., Conclusions: Instituting preoperative SARS-CoV-2 testing and maintaining high levels of compliance is possible in the midst of a fluctuating pandemic., Competing Interests: None, (© 2020 Published by Elsevier Inc.)

Introduction: Identifying predictors of patient outcomes evaluated over time may require modeling interactions among variables while addressing within-subject correlation. Generalized linear mixed models (GLMMs) and generalized estimating equations (GEEs) address within-subject correlation, but identifying interactions can be difficult if not hypothesized a priori . We evaluate the performance of several variable selection approaches for clustered binary outcomes to provide guidance for choosing between the methods., Methods: We conducted simulations comparing stepwise selection, penalized GLMM, boosted GLMM, and boosted GEE for variable selection considering main effects and two-way interactions in data with repeatedly measured binary outcomes and evaluate a two-stage approach to reduce bias and error in parameter estimates. We compared these approaches in real data applications: hypothermia during surgery and treatment response in lupus nephritis., Results: Penalized and boosted approaches recovered correct predictors and interactions more frequently than stepwise selection. Penalized GLMM recovered correct predictors more often than boosting, but included many spurious predictors. Boosted GLMM yielded parsimonious models and identified correct predictors well at large sample and effect sizes, but required excessive computation time. Boosted GEE was computationally efficient and selected relatively parsimonious models, offering a compromise between computation and parsimony. The two-stage approach reduced the bias and error in regression parameters in all approaches., Conclusion: Penalized and boosted approaches are effective for variable selection in data with clustered binary outcomes. The two-stage approach reduces bias and error and should be applied regardless of method. We provide guidance for choosing the most appropriate method in real applications., Competing Interests: The authors have no conflicts of interest to declare., (© The Association for Clinical and Translational Science 2020.)

RNA abasic sites and the mechanisms involved in their regulation are mostly unknown; in contrast, DNA abasic sites are well-studied. We found surprisingly that, in yeast and human cells, RNA abasic sites are prevalent. When a base is lost from RNA, the remaining ribose is found as a closed-ring or an open-ring sugar with a reactive C1' aldehyde group. Using primary amine-based reagents that react with the aldehyde group, we uncovered evidence for abasic sites in nascent RNA, messenger RNA, and ribosomal RNA from yeast and human cells. Mass spectroscopic analysis confirmed the presence of RNA abasic sites. The RNA abasic sites were found to be coupled to R-loops. We show that human methylpurine DNA glycosylase cleaves N-glycosidic bonds on RNA and that human apurinic/apyrimidinic endonuclease 1 incises RNA abasic sites in RNA-DNA hybrids. Our results reveal that, in yeast and human cells, there are RNA abasic sites, and we identify a glycosylase that generates these sites and an AP endonuclease that processes them., Competing Interests: The authors declare no competing interest., (Copyright © 2020 the Author(s). Published by PNAS.)

DNA polymerase (pol) β catalyzes two reactions at DNA gaps generated during base excision repair, gap-filling DNA synthesis and lyase-dependent 5´-end deoxyribose phosphate removal. The lyase domain of pol β has been proposed to function in DNA gap recognition and to facilitate DNA scanning during substrate search. However, the mechanisms and molecular interactions used by pol β for substrate search and recognition are not clear. To provide insight into this process, a comparison was made of the DNA binding affinities of WT pol β, pol λ, and pol μ, and several variants of pol β, for 1-nt-gap-containing and undamaged DNA. Surprisingly, this analysis revealed that mutation of three lysine residues in the lyase active site of pol β, 35, 68, and 72, to alanine (pol β KΔ3A) increased the binding affinity for nonspecific DNA ∼11-fold compared with that of the WT. WT pol μ, lacking homologous lysines, displayed nonspecific DNA binding behavior similar to that of pol β KΔ3A, in line with previous data demonstrating both enzymes were deficient in processive searching. In fluorescent microscopy experiments using mouse fibroblasts deficient in PARP-1, the ability of pol β KΔ3A to localize to sites of laser-induced DNA damage was strongly decreased compared with that of WT pol β. These data suggest that the three lysines in the lyase active site destabilize pol β when bound to DNA nonspecifically, promoting DNA scanning and providing binding specificity for gapped DNA., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article.

Background: Prior studies examining bleeding with uterine evacuation have focused on high-volume centers performing over 1100 procedures annually. The aim of this study was to examine associations between blood loss and patient and procedural characteristics in a center performing fewer than 50 procedures annually., Methods: This retrospective cohort study, with institutional review board approval, utilized procedural codes to identify patients undergoing uterine evacuation procedures between 14 weeks' and 24 weeks' gestational age across a 50-month period. The primary outcome was estimated blood loss; secondary outcomes were hemorrhage, transfusion and hospital re-admission. Associations between blood loss and other variables were examined using linear regression models., Results: Charts of 161 women met inclusion criteria. Median estimated blood loss was 400 mL (IQR 300 mL) with 37% of patients having blood loss of ≥500 mL. In univariate analyses, increased blood loss was associated with later gestational age (P <0.001) and pregnancy termination (P <0.001). In a multiple linear regression model, both remained significant. Each one-week increase in gestational age was associated with a 7.1% mean increase in estimated blood loss (95% CI 2.47% to 11.9%; P=0.003). Patients whose uterine evacuation was indicated for pregnancy termination had an 80.6% increase in blood loss compared with those with pre-operative fetal demise (95% CI 37.5% to 137.2%; P <0.001). Rates of peri-operative transfusion and re-admission for bleeding were <4%., Conclusion: While blood loss may be greater in low volume centers, our transfusion and re-admission rates were low following second trimester uterine evacuation., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Adenine frequently pairs with the Hoogsteen edge of an oxidized guanine base (8OG) causing G to T transversions. The (syn)8OG:dA base pair is indistinguishable from the cognant base pair and can be extended by DNA polymerases with reduced efficiency. To examine the structural basis of this reduced efficiency, we sought to obtain the structure of the "product" complex of DNA polymerase (pol) β with the (syn)8OG:dA base pair at the primer terminus by soaking the binary complex crystals with a hydrolysable dCTP analogue complementary to the template base G. Crystallographic refinement of the structure revealed that the adenine of the (syn)8OG:dA base pair had been expelled from the primer terminus and a dCMP was inserted opposite 8OG in a reverse orientation; another uninserted molecule of the analogue was bound to the templating base G. This leads to an abortive complex that could form the basis of oxidatively-induced pol β stalling.

Nucleotide excision repair (NER) removes helix-destabilizing adducts including ultraviolet (UV) lesions, cyclobutane pyrimidine dimers (CPDs), and pyrimidine (6-4) pyrimidone photoproducts (6-4PPs). In comparison with CPDs, 6-4PPs have greater cytotoxicity and more strongly destabilizing properties of the DNA helix. It is generally believed that NER is the only DNA repair pathway that removes the UV lesions as evidenced by the previous data since no repair of UV lesions was detected in NER-deficient skin fibroblasts. Topoisomerase I (TOP1) constantly creates transient single-strand breaks (SSBs) releasing the torsional stress in genomic duplex DNA. Stalled TOP1-SSB complexes can form near DNA lesions including abasic sites and ribonucleotides embedded in chromosomal DNA. Here we show that base excision repair (BER) increases cellular tolerance to UV independently of NER in cancer cells. UV lesions irreversibly trap stable TOP1-SSB complexes near the UV damage in NER-deficient cells, and the resulting SSBs activate BER. Biochemical experiments show that 6-4PPs efficiently induce stable TOP1-SSB complexes, and the long-patch repair synthesis of BER removes 6-4PPs downstream of the SSB. Furthermore, NER-deficient cancer cell lines remove 6-4PPs within 24 h, but not CPDs, and the removal correlates with TOP1 expression. NER-deficient skin fibroblasts weakly express TOP1 and show no detectable repair of 6-4PPs. Remarkably, the ectopic expression of TOP1 in these fibroblasts led them to completely repair 6-4PPs within 24 h. In conclusion, we reveal a DNA repair pathway initiated by TOP1, which significantly contributes to cellular tolerance to UV-induced lesions particularly in malignant cancer cells overexpressing TOP1., Competing Interests: The authors declare no competing interest.

DNA replication and repair reactions involve the addition of a deoxynucleoside monophosphate onto a growing DNA strand with the loss of pyrophosphate. This chemical reaction is also reversible; the addition of pyrophosphate generates a deoxynucleoside triphosphate, thereby shortening the DNA by one nucleotide. The forward DNA synthesis and reverse pyrophosphorolysis reactions strictly require the presence of divalent metals, usually magnesium, at the reactive center as cofactors. The overall equilibrium enzymatic reaction strongly favors DNA synthesis over pyrophosphorolysis with natural substrates. The DNA polymerase β chemical reaction has been structurally and kinetically characterized, employing natural and chemically modified substrates. Substituting an imido-moiety (NH) for the bridging oxygen between Pβ and Pγ of dGTP dramatically decreased the overall enzymatic activity and resulted in a chemical equilibrium that strongly favors the reverse reaction (i.e., K ≪ 1). Using QM/MM calculations in conjunction with the utilization of parameters such as quantum mechanically derived atomic charges, we have examined the chemical foundation for the altered equilibrium with this central biological reaction. The calculations indicate that the rapid reverse reaction is likely due, in part, to the increased nucleophilicity of the reactive oxygen on the tautomeric form of imidodiphosphate., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 U.S. Government.)

Competing Interests: Declaration of Competing Interest None of the authors has a conflict of interest.

The Publisher regrets that this article is an accidental duplication of an article that has already been published in DNA Repair, 90C (2020) 102850, https://doi.org/10.1016/j.dnarep.2020.102850. The duplicate article has therefore been withdrawn. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/our-business/policies/article-withdrawal.

Formaldehyde (FA) is a simple biological aldehyde that is produced inside cells by several processes such as demethylation of DNA and proteins, amino acid metabolism, lipid peroxidation and one carbon metabolism (1-C). Although accumulation of excess FA in cells is known to be cytotoxic, it is unknown if an increase in FA level might be associated with mitochondrial dysfunction. We choose to use primary human fibroblasts cells in culture (foreskin, FSK) as a physiological model to gain insight into whether an increase in the level of FA might affect cellular physiology, especially with regard to the mitochondrial compartment. FSK cells were exposed to increasing concentrations of FA, and different cellular parameters were studied. Elevation in intracellular FA level was achieved and was found to be cytotoxic by virtue of both apoptosis and necrosis and was accompanied by both G2/M arrest and reduction in the time spent in S phase. A gene expression assessment by microarray analysis revealed FA affected FSK cells by altering expression of many genes including genes involved in mitochondrial function and electron transport. We were surprised to observe increased DNA double-strand breaks (DSBs) in mitochondria after exposure to FA, as revealed by accumulation of γH2A.X and 53BP1 at mitochondrial DNA foci. This was associated with mitochondrial structural rearrangements, loss of mitochondrial membrane potential and activation of mitophagy. Collectively, these results indicate that an increase in the cellular level of FA can trigger mitochondrial DNA double-strand breaks and dysfunction.

The human DNA polymerase (pol) β cancer variant K289M has altered polymerase activity in vitro , and the structure of wild-type pol β reveals that the K289 side chain contributes to a network of stabilizing interactions in a C-terminal region of the enzyme distal to the active site. Here, we probed the capacity of the K289M variant to tolerate strain introduced within the C-terminal region and active site. Strain was imposed by making use of a dGTP analogue containing a CF 2 group substitution for the β-γ bridging oxygen atom. The ternary complex structure of the K289M variant displays an alteration in the C-terminal region, whereas the structure of wild-type pol β is not altered in the presence of the dGTP CF 2 analogue. The alteration in the K289M variant impacts the active site, because the enzyme in the ternary complex fails to adopt the normal open to closed conformational change and assembly of the catalytically competent active site. These results reveal the importance of the K289-mediated stabilizing network in the C-terminal region of pol β and suggest an explanation for why the K289M cancer variant is deficient in polymerase activity even though the position 289 side chain is distal to the active site.

Free radical attack on C1' of deoxyribose forms the oxidized abasic (AP) site 2-deoxyribonolactone (dL). In vitro, dL traps the major base excision DNA repair enzyme DNA polymerase beta (Polβ) in covalent DNA-protein crosslinks (DPC) via the enzyme's N-terminal lyase activity acting on 5'-deoxyribose-5-phosphate residues. We previously demonstrated formation of Polβ-DPC in cells challenged with oxidants generating significant levels of dL. Proteasome inhibition under 1,10-copper-ortho-phenanthroline (CuOP) treatment significantly increased Polβ-DPC accumulation and trapped ubiquitin in the DPC, with Polβ accounting for 60-70 % of the total ubiquitin signal. However, the identity of the remaining oxidative ubiquityl-DPC remained unknown. In this report, we surveyed whether additional AP lyases are trapped in oxidative DPC in mammalian cells in culture. Poly(ADP-ribose) polymerase 1 (PARP1), Ku proteins, DNA polymerase λ (Polλ), and the bifunctional 8-oxoguanine DNA glycosylase 1 (OGG1), were all trapped in oxidative DPC in mammalian cells. We also observed significant trapping of Polλ, PARP1, and OGG1 in cells treated with the alkylating agent methylmethane sulfonate (MMS), in addition to dL-inducing agents. Ku proteins, in contrast, followed a pattern of trapping similar to that for Polβ: MMS failed to produce Ku-DPC, while treatment with CuOP or (less effectively) H 2 O 2 gave rise to significant Ku-DPC. Unexpectedly, NEIL1 and NEIL3 were trapped following H 2 O 2 treatment, but not detectably in cells exposed to CuOP. The half-life of all the AP lyase-DPC ranged from 15-60 min, consistent with their active repair. Accordingly, CuOP treatment under proteasome inhibition significantly increased the observed levels of DPC in cultured mammalian cells containing PARP1, Ku protein, Polλ, and OGG1 proteins. As seen for Polβ, blocking the proteasome led to the accumulation of DPC containing ubiquitin. Thus, the ubiquitin-dependent proteolytic mechanisms that control Polβ-DPC removal may also apply to a broad array of oxidative AP lyase-DPC, preventing their toxic accumulation in cells., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)